Hepatitis C Virus (HCV) infection is a growing worldwide concern. HCV infections are generally persistent and induce chronic liver disease, manifested in cirrhosis of the liver and hepatocellular carcinoma. HCV is the leading cause for liver transplantation in the United States. Worldwide, approximately one million new HCV infections are reported annually; in the United States alone, an estimated four million persons are infected and 30,000 new infections occur annually.
Currently, HCV is responsible for an estimated 8,000 to 10,000 deaths annually in the United States. Without the development of improved diagnostics and therapeutics, that number is expected to triple in the next 10 to 20 years (National Institutes of Health Consensus Development Conference Panel (1997) National Institutes of Health Consensus Development Conference Panel statement: “Management of Hepatitis C,” Hepatology 26(Suppl. 1):2S-10S).
The HCV genome is highly polymorphic, and a number of strains (termed genotypes and subtypes) have been characterized. The different viral types correlate with different disease outcomes and different responsiveness to therapeutic regimens. Knowing the viral genotype (and/or subtype) present in an infection provides the clinician with an important indicator for determining an optimal course of treatment for an infected patient. However, the development of diagnostic methods that can differentiate the ever-increasing number of known HCV types has become a challenge.
There is a need in the art to develop improved methods for HCV diagnostics. There is a need for improved methods that can distinguish the increasingly large number of known HCV genotypic isolates, including genotypic subtypes. Furthermore, there is also a need in the art for methods that can simultaneously genotype and quantitate (e.g., determine the viral load or copy number) an HCV in a sample. The present invention provides novel compositions and methods that meet these needs, as well as provides additional benefits.
Prior to a detailed description of the present invention, pertinent aspects of HCV nomenclature and biology are discussed below. These topics, required for understanding the invention, include discussion of the HCV genome, HCV typing nomenclature, clinical relevance of HCV typing and HCV typing methodologies.
HCV Genome
The HCV genome (see, FIG. 1) has a positive-sense single-stranded RNA genome approximately 10 kb in length with marked similarities to the genomes of members of the Pestivirus and Flavivirus genera. The original HCV isolate (HCV-1) had an approximately 9.4 kB genome containing a poly(A) tail at the 3′ end (Choo et al. (1991) “Genetic organization and diversity of the hepatitis C virus,” Proc. Natl. Acad. Sci. USA 88:2451-2455). The HCV-1 sequence contained a 5′ untranslated region (5′-UTR) of 341 bases, a long open reading frame encoding a polyprotein of 3,011 amino acids, and a 3′ untranslated region (3′-UTR) of about 27 bases. See the schematic of the HCV genome and polyprotein in FIG. 1.
The viral RNA genome is translated by the host translation apparatus as a single polyprotein product, which is then subjected to proteolytic processing to produce the viral proteins. The length of the open reading frame (ORF) of each genotype is characteristically different. For example, the open reading frame in type 1 isolates is approximately 9,400 ribonucleotides in length, while that of type 2 isolates is typically 9,099 nucleotides and that of type 3 isolates is typically 9,063 nucleotides (Bukh et al. (1995) “Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes,” Semin. Liver Dis., 15:41-63).
The HCV genomic structure/organization is most similar to that of the family Flaviviridae. Consistent with the known functions of most flavivirus proteins, the N-terminal HCV proteins are likely structural (including the C (capsid/core), E1 and E2 envelope proteins) and the C-terminal non-structural proteins, including NS2 (metalloprotease), NS3 (serine-protease/helicase), NS4 and NS5 (NS5B RNA polymerase) are believed to function in viral replication. A schematic view showing organization of the HCV RNA genome and encoded polypeptides is provided in FIG. 1.
Following identification and characterization of the prototypical HCV isolate (now termed HCV 1a), other isolates from around the world were (and continue to be) identified. Sequence comparisons reveal that these unique isolates can differ from each other by as much as 35% nucleotide non-identity over the full length of the HCV genome (Okamoto et al. (1992) Virology 188:331-341). Sequence variability is observed throughout the viral genome, with some regions showing more variability than others. For example, generally high sequence conservation is observed in the 5′-UTR region; conversely, some regions, including the envelope (E) region, show hypervariable nucleotide sequences.
HCV Typing Nomenclature
An understanding of HCV typing nomenclature is required prior to discussion of the present invention. Historically, investigators have used several classification systems and nomenclatures to characterize the various HCV strains, resulting in confusion in the scientific literature. A consensus HCV genotype/subtype nomenclature system has now been adopted (Simmonds et al. (1994) Letter, Hepatology 19:1321-1324; see also, Zein (2000) “Clinical Significance of Hepatitis C Virus Genotypes,” Clinical Microbiol. Reviews 13(2):223-235; Maertens and Stuyver (1997) “Genotypes and Genetic Variation of Hepatitis C Virus,” p. 182-233, In Harrison, and Zuckerman (eds.), The Molecular Medicine of Viral Hepatitis, John Wiley & Sons, Ltd., Chichester, England). According to this system, HCV isolates are classified on the basis of nucleotide sequence divergence into major genetic groups designated as genotypes. These genotypes are numbered (in Arabic numerals), generally in the order of their discovery. HCV strains that are more closely related to each other within a genotype are designated as subtypes, which are assigned lowercase letters, generally in the order of their discovery. Genetic variants found within an individual isolate are termed quasispecies. Quasispecies of HCV result presumably from the accumulation of mutations during viral replication in the host.
The degree of relatedness between any two HCV isolates can be quantitated, for example, by determining the percentage of nucleotide identity between the two genomes over the full length of the genome. One example of this relatedness analysis, and how the nomenclature is used to reflect viral isolate relatedness, is shown in FIG. 2 (adapted from Zein (2000) “Clinical Significance of Hepatitis C Virus Genotypes,” Clinical Microbiol. Reviews 13(2):223-235). Using the nomenclature proposed by Simmonds et al. (1994, Letter, Hepatology 19:1321-1324), the increasing degree of interrelatedness between genotypes, subtypes and quasispecies can be observed in the percentage of nucleotide sequence identity over the complete genome. The table in FIG. 2 reflects the proposal that HCV isolates that are quasispecies share the greatest degree of relatedness, and isolates of the same subtype within a genotype share greater sequence identity than isolates of different subtypes also within that genotype.
Alternatively, relatedness between HCV isolates can be quantitated by examining genomic identity over a smaller domain of the genome, as shown, for example, in FIG. 3. This comparison uses a 222 nucleotide segment derived from the viral NS5 open reading frame (nucleotide positions 7975-8196 in the prototype HCV type 1a isolate). This comparison of sequence identity also supports the proposal of Simmonds et al. (1994, Letter, Hepatology 19:1321-1324) that HCV isolates of one subtype are more closely related to other subtypes of that same genotype, than to isolates from any other genotype.
Currently, eleven (11) HCV genotypes are recognized worldwide. However, there is published suggestion that the evolutionary (phylogenetic) relatedness between different genotypes should be reexamined, and the number of recognized genotypes into which HCV isolates are classified/assigned should be reassessed. Some reports suggest that subsets of HCV genotypes are more closely related to each other than to other more distantly related genotypes, which should be reflected in a modified HCV nomenclature. It is suggested that the 11 genotypes can be regrouped into six HCV clades. The grouping of clades reflects phylogenetic relationships between the genotypes, where genotypes 1, 2, 4 and 5 all represent distinct clades, but where genotypes 3 and 10 are placed into a single lade 3, and genotypes 6, 7, 8, 9 and 11 are placed into a single lade 6 (Robertson et al., (1998) Arch. Virol., 143(12):2493-2503; Zein (2000) “Clinical Significance of Hepatitis C Virus Genotypes,” Clinical Microbiol. Reviews 13(2):223-235).
Approximately 78 HCV subtypes encompassing all 11 genotypes are known worldwide. A summary of some of these subtypes is shown in FIG. 4. This table provides a listing of some (but not all) HCV types, especially those subtypes that appear to be frequent clinical isolates. The names of prototypical and/or representative isolates known in the art are provided.
Many HCV isolates have been sequenced in their entirety. FIG. 5 provides a table showing the consensus sequences of a 33 nucleotide domain in the 5′-UTR of some of the clinically relevant HCV isolates. Nucleotide positions that are identical to the HCV type 1a nucleotide sequence are shown with a dash. Nucleotide positions that differ from the HCV type 1a are shown with the nucleotide change.
All references to HCV genotypes, subtypes and quasispecies herein are in accordance with the system described by Simmonds et al., 1994, (Letter, Hepatology 19:1321-1324), and also described in, for example, Zein (2000) “Clinical Significance of Hepatitis C Virus Genotypes,” Clinical Microbiol. Reviews 13(2):223-235; Maertens and Stuyver (1997) “Genotypes and Genetic Variation of Hepatitis C Virus,” p. 182-233, In Harrison, and Zuckerman (eds.), The Molecular Medicine of Viral Hepatitis, John Wiley & Sons, Ltd., Chichester, England.
Clinical Relevance of HCV Typing
The typing of an HCV infection in a patient remains an important prognosticator for the aggressiveness of the infection, as well as the potential for the infection to respond to various therapeutic regimens. HCV genotype 1 represents a more aggressive strain and one that is less likely to respond to alpha interferon (INF-α) treatment (and combination therapies with ribavirin) than HCV genotype 2 or 3 (Nolte, “Hepatitis C Virus Genotyping: Clinical Implications and Methods,” Molecular Diagnosis 6(4):265-277 [2001]; Dufour, “Hepatitis C: Laboratory Tests for Diagnosis and Monitoring of Infection,” Clinical Laboratory News, November 2002, p. 10-14; Pawlotsky “Use and Interpretation of Hepatitis C Virus Diagnostic Assays,” Clinics in Liver Disease, Vol. 7, Number 1 [February 2003]). The goal in typing an HCV infection is frequently to identify patients infected with HCV genotype 1 as opposed to those infected with other HCV types. Furthermore, with the identification of an expanding list of known HCV subtypes, there is a need in the art for simple HCV typing methods that can distinguish the complexity of HCV phylogeny for both clinical and research purposes. There is also a need in the art for HCV typing methods that simultaneously provide HCV load information (e.g., copy number and viral genome quantitation).
Substantial regional differences exist in the distribution of the HCV types. HCV subtypes 1a and 1b are the most common subtypes in the United States and Europe. In Japan, subtype 1b is responsible for up to 73% of cases of HCV infection. Although HCV subtypes 2a and 2b are relatively common in North America, Europe, and Japan, subtype 2c is found commonly in northern Italy. HCV genotype 3a is particularly prevalent in intravenous drug abusers in Europe and the United States. HCV genotype 4 appears to be prevalent in North Africa and the Middle East, and genotypes 5 and 6 seem to be confined to South Africa and Hong Kong, respectively. HCV genotypes 7, 8, and 9 have been identified only in Vietnamese patients, and genotypes 10 and 11 were identified in patients from Indonesia. (see, Nolte, “Hepatitis C Virus Genotyping: Clinical Implications and Methods,” Molecular Diagnosis 6(4):265-277 [2001]; Pawlotsky “Hepatitis C Virus Genetic Variability: Pathogenic and Clinical Implications,” Clinics in Liver Disease, Vol. 7, Number 1 [February 2003]; Zein “Clinical Significance of Hepatitis C Virus Genotypes,” Clinical Microbiol. Reviews 13(2):223-235 [2000]).
Because of the geographic clustering of distinct HCV genotypes and subtypes, HCV typing can also be a useful epidemiologic marker in tracing the source of an HCV outbreak in a given population. For example, HCV typing was used to trace the source of HCV infection in a group of Irish women to contaminated anti-D immunoglobulins (Power et al., (1995) “Molecular epidemiology of an outbreak of infection with hepatitis C virus in recipients of anti-D immunoglobulin,” Lancet 345:1211-1213). Additional examples of using HCV typing as an epidemiological marker are also known (see, for example, Hohne et al., (1994) “Sequence variability in the env-coding region of hepatitis C virus isolated from patients infected during a single source outbreak,” Arch. Virol., 137:25-34; and Bronowicki et al., (1997) “Patient-to-patient transmission of hepatitis C virus during colonoscopy,” N. Engl. J. Med., 337:237-240).
HCV Typing Methodologies
The HCV isolate found in any given infection varies by differences in geographical strain distribution, disease outcome, and response to anti-HCV therapy. Reliable methods for determining HCV genotype are an important clinical test. Furthermore, with the identification of numerous and distinct HCV species, it is important that HCV typing methods have the ability to distinguish numerous HCV types (genotypes and subtypes). For example, it is useful for an HCV genotyping test to be able to distinguish among at least five or more genotypes. Alternatively, it is useful for an HCV typing test to be able to distinguish among at least six or more subtypes. Nucleic acid-based methods for HCV typing are summarized below.
Nucleotide Sequencing
The reference standard for HCV genotyping and subtyping is nucleotide sequencing of an amplicon derived from the HCV genome by RT-PCR of HCV genomic RNA (e.g., from a clinical specimen from a patient) followed by phylogenetic assignment. However, direct sequencing is impractical due to low throughput (even with the introduction of automated sequencing apparatus using non-radioactive reagents) and the requirement for specialized equipment. Furthermore, using sequencing methodologies for genotyping and subtyping in cases of mixed infections can result in ambiguous results.
PCR-Based HCV Genotyping
Some typing methods use PCR reamplification using type-specific PCR primers. Typing is achieved by a primary PCR amplification with universal consensus primers (i.e., primers that will generate an HCV genomic amplicon regardless of the HCV type) followed by a nested PCR with the type-specific primers, for example, type-specific primers within the core region. These assays require multiple sets of PCR primers to generate sufficient type-specific PCR amplicons to make a genotype/subtype assignment. These methods have the drawback that they require multiple sets of PCR primers to accomplish the HCV typing, and often lack sensitivity and specificity (Xavier and Bukh (1998) “Methods for determining the hepatitis C genotype,” Viral Hepatitis Rev., 4:1-19; Zein (2000) “Clinical Significance of Hepatitis C Virus Genotypes,” Clinical Microbiol. Reviews 13(2):223-235; Okamoto et al., (1992) “Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources,” J. Gen. Virol., 73:673-679; Widell et al. (1994) “Genotyping of hepatitis C virus isolates by a modified polymerase chain reaction assay using type specific primers: epidemiological applications,” J. Med. Virol., 44:272-279).
Hybridization-Based HCV Genotyping
The typing of HCV isolates can be achieved by using multiple type-specific hybridization probes. This viral typing uses a primary PCR amplification using universal primers followed by hybridization with type-specific hybridization probes. This hybridization with the type-specific probes is done in fixed hybridization conditions, and the presence or absence of a hybridization complex(es) under the given hybridization conditions is scored. Any one probe in the assay is unable to definitively distinguish from among multiple genotypes/subtypes, thus necessitating the use of multiple probes to make a genotype/subtype assignment. This approach suffers from the drawback of requiring multiple probes for use in the HCV typing process.
One application of the HCV type-specific hybridization assay is the line-probe assay (LiPA), as described in various sources (Stuyver et al., (1993) “Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay,” J. Gen. Virol., 74:1093-1102; Stuyver et al., (1994) Proc. Natl. Acad. Sci. USA 91:10134-10138; Andonov and Chaudhary (1995) Jour. Clin. Microbiol., 33(1):254-256; Stuyver et al., (1996) Jour. Clin. Microbiol., 34(9):2259-2266; Stuyver et al., (2000) Jour. Clin. Microbiol., 38(2):702-707; and reviewed in, e.g., Maertens and Stuyver (1997) “Genotypes and genetic variation of hepatitis C virus,” p. 182-233, In Harrison and Zuckerman (eds.), The Molecular Medicine Of Viral Hepatitis, John Wiley & Sons, Ltd., Chichester, England). A commercial kit incorporating this technology is produced by Innogenetics (Zwijnaarde, Belgium; see U.S. Pat. No. 6,548,244, issued Apr. 15, 2003 to Maertens et al., entitiled “PROCESS FOR TYPING HCV ISOLATES”; Published PCT International Application No. WO96/13590, published May 9, 1996, by Maertens and Stuyver, entitled “NEW SEQUENCES OF HEPATITIS C VIRUS GENOTYPES AND THEIR USE AS PROPHYLACTIC, THERAPEUTIC AND DIAGNOSTIC AGENTS”; and Published PCT International Application No. WO94/25601, published Nov. 10, 1994, by Maertens and Stuyver, entitled “NEW SEQUENCES OF HEPATITIS C VIRUS GENOTYPES AND THEIR USE AS THERAPEUTIC AND DIAGNOSTIC AGENTS”). The line-probe assay uses multiple type-specific probes (as many as 21 probes) immobilized onto a substrate (a test strip) in a dot-blot or slot-blot type of assay. An HCV amplicon derived from the HCV 5′-UTR region generated from a clinical specimen is simultaneously hybridized to the various probes under static hybridization conditions, and the resulting pattern of hybridization complexes reveals the virus type.
This line-probe assay suffers from the drawback of requiring the use of multiple probes (indeed, as many as 21 probes) to determine the HCV genotype and/or subtype of an HCV in a sample, as any one probe in the assay is unable to make a genotypic assignment.
Some reports use HCV typing methods that utilize one probe (or a small number of probes) to classify an HCV infection into one of a few subtypes. The probes used in these reports are not “type-specific,” in that they can hybridize to multiple genotypes/subtypes by manipulating the hybridization conditions. However, these types of probes reported in the art (see, e.g., Schroter et al., (2002) Jour. Clin. Microbiol., 40(6):2046-2050; Bullock et al., (2002) Clinical Chemistry 48(12):2147-2154) are limited in the number of genotypes/subtypes they can differentiate.
Endonuclease Cleavage (RFLP)-Based HCV Genotyping
Typing of HCV has also been attempted by a variation of the traditional restriction fragment length polymorphism (RFLP) assay. This HCV assay uses digestion of a universal PCR amplicon with restriction endonucleases that recognize genotype-specific cleavage sites (see, e.g., Nakao et al., (1991) “Typing of hepatitis C virus genomes by restriction fragment length polymorphism,” J. Gen. Virol., 72:2105-2112; Murphy et al., (1994) Letter, J. Infect. Dis., 169:473-475). Type-specific restriction sites are known to occur in the NS5 and the 5′-UTR domains. The use of this assay for genotyping/subtyping is limited due to the limited number of polymorphic loci that result in changes in restriction sites.
HCV Typing Challenges
Determining an HCV virus type in a sample is an important clinical tool. One technique for making an HCV type determination is to characterize the melting temperatures of hybridization complexes formed with an HCV-specific probe using known HCV types and the experimental HCV sample. Ideally, an HCV typing probe in a complex with an HCV genomic sequence (or derivative of and/or portion of an HCV genomic sequence) will yield a unique Tm value corresponding to a specific HCV type, and thus the assignment of a particular HCV type is made on the basis of the experimentally observed Tm value.
However, as the numbers of known HCV types increases and more quasispecies are identified, the use of a single probe to make an HCV type determination becomes more challenging. This is illustrated in FIG. 8. In this figure, a hypothetical HCV Typing Probe A forms complexes with HCV genotypes 1 through 6. These complexes are each characterized by a range of Tm values, as opposed to one absolute value. A number of variables contribute to this range in Tm values, including but not limited to (i) HCV sequence variants such as quasispecies, (ii) the particular melting analysis reaction conditions (e.g., salt concentrations and the concentrations of the nucleic acids in the hybridization reactions), and (iii) the accuracy and precision of the melting analysis instrumentation, including the thermocycling device and the detector (e.g., the spectrophotometer). As shown in FIG. 8, it is possible that the Tm value range for any one HCV/probe combination overlaps with or can not be distinguished from the Tm values for another HCV type using that same probe. This situation makes a definitive determination of an HCV type impossible or ambiguous.
The present invention provides compositions and methods for HCV typing, and furthermore, provides compositions and methods for HCV typing that have advantages over other HCV typing methods known in the art. The compositions and methods for HCV typing described herein overcome the limitations in HCV typing as illustrated in FIG. 8. The invention provides methods for typing an HCV isolate, where the methods use a multidimensional analysis where combinations of HCV typing probes provide unique and complementary information on individual HCV types and are able to distinguish at least five HCV types (genotypes and/or subtypes). The process of multidimensional typing provides multiple datapoints to characterize each HCV type, increasing the accuracy and robustness of typing determination, while simultaneously providing a solution for discrepancies arising from intra-genotype variance. Furthermore, the invention provides methods that can simultaneously type an HCV in a sample as well as quantitate the HCV genomic material in the sample (e.g., determine the viral load or copy number). The compositions and methods taught by the present invention also provide other advantages, which will be apparent upon reading the description of the invention.